350 Maunderstions can be varied to give the optimal reaction with a particular sub-
strate. For example, when kinasing blunt or 5'-recessed termini, higher
concentrations of ATP and enzyme are necessary (27), and additional
buffer components such as PEG may be beneficial (44). Several reac-
tion protocols are given in Section 4.4.
A number of points are worthy of note. First, because of the extreme
inhibition of T4 polynucleotide kinase by ammonium salts, the prepa-
ration of the polynucleotide substrate should not involve precipitation
with ammonium acetate prior to the kinasing step. Second, all reaction
buffers used should be Tris- or imidazole-based, and not contain inor-
ganic phosphate; otherwise inhibition will be observed again. Finally,
the target molecule should be rigorously purified from small mol-wt
fragments, since these would contribute a large number of 5' termini,
which could compete in the reaction.
After the kinasing reaction, subsequent purification depends on the
use to which the phosphorylated polynucleotide will be put. If it is to
be used as a hybdridization probe, further purification is not necessary
unless problems with background noise have been encountered. Puri-
fication by repeated ethanol precipitation may be sufficient. When
working with oligonucleotides of <18 bases in length, precipitation
with cetylpyridinium bromide (27,45) must be employed. If the mol-
ecule is to be used in further manipulations, it may need more rigorous
purification by, e.g., column chromatography, spin dialysis, or poly-
acrylamide gel electrophoresis.
4.4. Reaction Protocols
4.4.1. Materials Required- Suitably prepared 5'-OH DNA, RNA, or oligonucleotides.
- Reaction buffers (use one or the other):
a. For kinasing, 10X Tris kinase buffer: 500 mM Tris-HCl, pH 7.6, 100
mM MgCl 2, 50 mM DTT, 1 mM Spermidine, and 1 mM EDTA.
b. For kinasing or phosphate exchange, 10X imidazole buffer: 500 mM
imidazole-Cl, pH 6.4; 180 mM MgC 12; 50 mM DTT; 1 mM Spermi-
dine; and 1 mM EDTA. - •-32P-ATP (3000 or 5000 Ci/mmol).
- T4 polynucleotide kinase (10 U/lxL).
- 500 mM EDTA, pH 8.0.
- Sterile distilled water.
- Phenol/chloroform and chloroform for extraction.